The omega-3 fatty acid receptor (O3FAR1), also commonly referred to as GPR120, is a G protein-coupled receptor (GPCR) that has been shown to be agonized by long chained unsaturated fatty acids, specifically, those belonging to the omega-3 fatty acid (O3FA) family. This receptor has generated considerable interest due to its ability to stimulate intestinal secretion of the incretin hormone glucagon-like-peptide-1 (GLP-1), which stimulates pancreatic b-cells leading to insulin secretion and subsequently, decreases in blood glucose. In addition to this effect, it has recently been demonstrated that agonism of O3FAR1 leads to profound anti- inflammatory effects via inhibition of the NF-KB and JNK proinflammatory pathways, which when otherwise activated in macrophages, lead to insulin resistance, weight gain, and type 2 diabetes. Our laboratory has revealed that two human O3FAR1 isoforms, O3FAR1-long and short, exist as a consequence of alternative mRNA splicing and our results suggest that these isoforms are differentially regulated by receptor phosphorylation. Our preliminary data also show that G protein-coupled receptor kinase-6 (GRK6) is responsible for homologous (agonist-induced) phosphorylation of O3FAR1 isoforms. Importantly, the antidiabetic effects of O3FAR1 have been shown to be dependent on b-arrestin-2 partner proteins, which are recruited only to GRK-phosphorylated receptors, suggesting that receptor phosphorylation drives O3FAR1 antidiabetic activity. Additionally, our results demonstrate that basal phosphorylation of O3FAR1, which regulates receptor expression and agonist sensitivity, is mediated by protein kinase C (PKC), and that there are differential affects of PKC on the two O3FAR1 isoforms. We propose two specific aims that will provide structural guidance on mechanisms of O3FAR1 activation and signal regulation, and will also provide a better understanding of signaling differences between the two O3FAR1 isoforms. In specific aim one, we will localize the specific sites of GRK and PKC mediated phosphorylation of O3FAR1 isoforms using a proteomic- driven approach. In specific aim two, we will assess the functional significance of these phosphorylation mechanisms with respect to their ability to regulate receptor interactions with b-arrestin-2, activate the Gaq/11- phospholipase C/Ca+2 signaling pathway, secrete GLP-1, and produce anti-inflammatory effects via inhibition of JNK and NF-KB pathways. Collectively, these results will provide structural insight into mechanisms of phospho-regulation of all known O3FAR1 functions. Since research activities at Mercer University's College of Pharmacy and Health Sciences are fully dependent on student integration, this project will allow for incorporation of graduate (PhD), professional (PharmD), and undergraduate students within an established and significant research project, and will greatly strengthen the research environment, consistent with the goals of the AREA funding mechanism.